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Method and composition for building HBV transgenic mouse model, and applications of HBV transgenic mouse model

A technology of transgenic mice and compositions, which can be applied to other methods, applications, genetic engineering and other directions of inserting foreign genetic materials, can solve the problems of low transgenic efficiency, gene silencing, restricted recombinant ES cell preparation, etc., and achieve increased expression stability. Sex, expression stabilization effect

Pending Publication Date: 2020-10-16
广州华腾生物医药科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the transgenic efficiency of microinjection mice is low, and HBV is randomly integrated, which is prone to gene silencing or loss in the genetic process of offspring
The positive rate of transgenic mice by gene targeting is higher than that by microinjection, but it is limited by the preparation of recombinant ES cells in the early stage

Method used

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  • Method and composition for building HBV transgenic mouse model, and applications of HBV transgenic mouse model
  • Method and composition for building HBV transgenic mouse model, and applications of HBV transgenic mouse model
  • Method and composition for building HBV transgenic mouse model, and applications of HBV transgenic mouse model

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Construction of plasmids expressing sgRNA

[0030] 1. sgRNA design

[0031] The sequence information of sgRNA is shown in Table 1 below:

[0032] Table 1 sgRNA design information

[0033]

[0034] 2. Construction of U6-Roas26-sgRNA

[0035] ① Dilute the synthesized sgRNA oligonucleotides to 10 μM, prepare the system according to Table 2, and perform programmed annealing on a PCR machine to form double-stranded DNA containing BbsI cohesive ends. The reaction procedure was heating at 98°C for 10 min, and cooling to room temperature naturally.

[0036]

[0037]

[0038] Table 2 sgRNA annealing reaction system

[0039] components Volume (uL) sgRNA-S 10 sgRNA-R 10 NEB buffer 3(10×) 5 wxya 2 o

25 Total 50

[0040] ② Linearization of U6-sgRNA expression vector: U6-sgRNA expression vector was digested with BbsI restriction endonuclease, and the enzyme digestion reaction system is shown in Table 3.

[0041] Table 3...

Embodiment 2

[0064] homology arm construction

[0065] Use PCR to amplify the mouse albumin promoter sequence, connect it into the PGEM-T vector, and then connect the promoter sequence and 1.3 copies of the whole HBV genome into pcDNA3.1 to form the homology arm expression plasmid pcDNA3.1-1.3 HBV-Donor.

[0066] HBV genome sequence (SEQ ID NO.4):

[0067] aattccacaaccttccaccaaactctgcaagatcccagagtgagaggcctgtatttccctgctggtggctccagttcaggaacagtaaaccctgttctgactactgcctctcccttatcgtcaatcttctcgaggattggggaccctgcgctgaacatggagaacatcacatcaggattcctaggaccccttctcgtgttacaggcggggtttttcttgttgacaagaatcctcacaataccgcagagtctagactcgtggtggacttctctcaattttctagggggaactaccgtgtgtcttggccaaaattcgcagtccccaacctccaatcactcaccaacctcttgtcctccaacttgtcctggttatcgctggatgtgtctgcggcgttttatcatcttcctcttcatcctgctgctatgcctcatcttcttgttggttcttctggactatcaaggtatgttgcccgtttgtcctctaattccaggatcctcaacaaccagcacgggaccatgccggacctgcatgactactgctcaaggaacctctatgtatccctcctgttgctgtaccaaaccttcggacggaaattgcacctgtattcccatcccatcatcctgggctttcggaaaattcctatgg...

Embodiment 3

[0075] Construction of transgenic ES cell lines

[0076] According to the results of the sgRNA activity detection experiment, the pU6-sgRNA with the highest activity on the target site was selected for subsequent experiments.

[0077] 1. Transfection and monoclonal screening

[0078] ① ES cells were co-transfected with 1 μg pU6-sgRNA, 1 μg pCas9 and 1 μg linearized pcDNA3.1-1.3HBV-Donor by electroporation, and pEGFR-N2 plasmid was set as the control group.

[0079] ② After 48 hours of transfection, add G418 screening medium (pre-test to determine drug screening concentration), observe and record the cell state every day, and replace fresh screening medium containing G418 every other day.

[0080] ③ After 7-10 days of G418 screening, almost all negative cells died. After the cells to be screened proliferated in large quantities, some cells were taken for limiting dilution and inoculated into 10 cm cell culture dishes so that there were about 30-40 cells in each dish, and the ...

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Abstract

The invention discloses a method and composition for building a HBV transgenic mouse model, and applications of the HBV transgenic mouse model, and relates to the technical field of biology. The disclosed method adopts a CRISP / Cas9 system to insert a HBV expression cassette at the Rosa26 locus in a mouse genome. A mouse model capable of continuously and stability HBV can be obtained by adopting the method; a HBV gene is inserted at the Rosa26 locus, so that HBV expression stability can be enhanced; mouse albumin promoters are adopted to drive expression, so that an exogenous HBV gene can be fixedly expressed in mouse liver, and the situations that HBV is persistently infected and expressed in human liver can be simulated; and the model can provide reliable model foundation for researchingHBV infection diseases and screening corresponding treatment drugs.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method, composition and application for constructing an HBV transgenic mouse model. Background technique [0002] Currently, HBV-related mouse models mostly use HBV plasmid hydrodynamic injection mouse models or AAV-HBV infection models. Although the preparation process of these models is relatively simple, due to the transient infection, the expression level of HBV in the mice is unstable, and the inter-individual differences are large, which cannot meet the needs of long-term observation and research. [0003] In addition, pronuclear microinjection and gene targeting techniques are often used in HBV transgenic mouse models. Pronuclear microinjection is to inject HBV DNA fragments directly into the pronuclei of fertilized eggs obtained by superovulation, and take the fertilized eggs that survived the injection and transplant them into the fallopian tubes of pseudopregnant mother ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/90C12N15/51A01K67/027
CPCA01K67/0278A01K2217/072A01K2227/105A01K2267/0337C07K14/005C12N15/907C12N2730/10122
Inventor 邹淑香谢水林邹芬陈莹莹黄黎珍游凯欣姚林敏
Owner 广州华腾生物医药科技有限公司
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